Nature - USA (2020-05-14)

(Antfer) #1

Methods


Data reporting
No statistical methods were used to predetermine sample size. The
experiments were not randomized and the investigators were not
blinded to allocation during experiments and outcome assessment.


Plasmids
SARS-CoV-2 spike (GenBank accession number QHD43416.1), SARS-CoV
spike (GenBank accession number AFR58740.1), RaTG13 spike (GenBank
accession number QHR63300.2) and ACE2 (GenBank accession number
NM_021804) were all synthesized (GenScript Biotech). SARS-CoV-2,
SARS-CoV, chimeric RBDs (see Extended Data Fig. 1 for residue ranges
of RBDs) and ACE2 ectodomain (residues 1–615) were subcloned into
pFastBac vector (Life Technologies) with a N-terminal honeybee melit-
tin signal peptide and a C-terminal His 6 -tag. The ACE2 ectodomain
(residues 1–615) with a C-terminal Fc-tag was also constructed.


Protein expression and purification
All of the proteins were prepared from Sf9 insect cells using the
Bac-to-Bac system (Life Technologies) as previously described^3. In
brief, the His 6 -tagged proteins were collected from cell culture medium,
purified using a Ni-NTA column, purified further using a Superdex200
gel filtration column (GE Healthcare) and stored in a buffer containing
20 mM Tris pH 7.2 and 200 mM NaCl. The Fc-tagged protein was purified
in the same way as the His 6 -tagged proteins, except that the protein A
column replaced the Ni-NTA column in the procedure.


Crystallization and structure determination
To purify the RBD–ACE2 complex, ACE2 and RBD were incu-
bated together, and the complex was purified using Superdex200
gel-filtration chromatography. RBD–ACE2 crystals were grown in sitting
drops at room temperature over wells containing 100 mM Tris (pH 8.5),
18–20% PEG 6000 and 100 mM NaCl. Crystals were soaked briefly in
100 mM Tris (pH 8.5), 30% PEG 6000, 100 mM NaCl and 30% ethylene
glycol before being flash-frozen in liquid nitrogen. X-ray diffraction
data were collected at the Advanced Photon Source beamline 24-ID-E.
The structure was determined by molecular replacement using the
structure of SARS-CoV RBD complexed with ACE2 as the search tem-
plate (Protein Data Bank (PDB) 2AJF). Structure data and refinement
statistics are shown in Extended Data Table 1.


Protein–protein binding assay
The SPR assays using a Biacore 2000 system (GE Healthcare) were
carried out as described previously^12. In brief, different RBDs were
covalently immobilized to a CM5 sensor chip through their amine
groups (GE Healthcare). The running buffer contained 10 mM HEPES
pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.05% Tween-20. Serial dilutions
of purified recombinant ACE2 were injected ranging in concentration
from 5 to 80 nM for the SARS-CoV-2 RBD and chimeric RBD, and from
20 to 320 nM for the SARS-CoV RBD. The resulting data were fit to a
1:1 binding model using Biacore Evaluation Software (GE Healthcare).
The protein pull-down assay was performed using a Dynabeads
His-Tag Isolation and Pull-down kit (Invitrogen) and a Dynabeads Pro-
tein A for Immunoprecipitation kit (Invitrogen) according to the manu-
facturers’ manual. In brief, 150 μl indicated Dynabeads were washed
with phosphate-buffered saline (PBS) and incubated with either 5 μg
ACE2–His 6 (ACE2 with a C-terminal His 6 -tag) or 5 μg ACE2–Fc (ACE2 with
a C-terminal Fc-tag) on a roller at room temperature for 30 min. After
incubation, ACE2-bound beads were washed three times with 1 ml PBST
buffer (PBS and 0.05% Tween-20) on a roller for 10 min and then were
aliquoted into different tubes for use. To prepare the cell-associated
coronavirus spike protein, HEK293T cells were transfected with
a pcDNA3.1(+) plasmid encoding coronavirus spike (containing a
C-terminal C9-tag); 48 h after transfection, the spike-expressing cells


were lysed using a sonicator in immunoprecipitation assay buffer
(20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA and 1% Triton X-100,
supplemented with protease inhibitors) and centrifuged at 12,000g for
2 min. The supernatants (containing solubilized SARS-CoV-2 spike) were
transferred to mix with the ACE2-bound beads in 2-ml tubes separately
(spike was in excess of ACE2). After a 1-h incubation on a roller at room
temperature, beads were washed three times with PBST buffer and the
bound proteins were eluted using elution buffer (300 mM imidazole,
50 mM sodium phosphate pH 8.0, 300 mM NaCl, 0.01% Tween-20 for
ACE2–His 6 -bound beads; 0.1 M citric acid pH 2.7 for ACE2–Fc-bound
beads). The samples were then subjected to SDS–PAGE and analysed
by western blotting using an anti-C9 tag antibody.

Coronavirus-spike-mediated pseudovirus entry assay
The pseudovirus entry assay was performed as described previously^21. In
brief, HEK293T cells were co-transfected with a luciferase-expressing HIV-1
genome plasmid (pNL4-3.luc.RE) and a plasmid encoding SARS-CoV-2
spike or RaTG13 spike. Pseudoviruses were collected 72 h after transfec-
tion, and were used to enter recipient cells (HEK293T cells exogenously
expressing ACE2). After incubation of pseudoviruses with recipient cells
at 37 °C for 6 h, the medium was changed and cells were incubated for
an additional 60 h. Cells were then washed with PBS buffer and lysed.
Aliquots of cell lysates were transferred to Optiplate-96 (PerkinElmer),
followed by the addition of luciferase substrate. Relative light units were
measured using an EnSpire plate reader (PerkinElmer). All measurements
were carried out on at least three independent biological samples.

Analyses of protein contact residues and protein buried surface areas
Protein contact residues were analysed using the LigPlot+ program
(v.1.4.5) (https://www.ebi.ac.uk/thornton-srv/software/LigPlus/).
Protein buried surface areas were analysed using PDBePISA tool (http://
pdbe.org/pisa/).

Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.

Data availability
Coordinates and structure factors have been deposited to the Protein
Data Bank with accession number 6VW1.


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Acknowledgements This work was supported by NIH grants R01AI089728 and R01AI110700
(to F.L.) and R35GM118047 (to H.A.) and is based on research conducted at the Northeastern
Collaborative Access Team beamlines, which are supported by NIH grants P30GM124165 and
S10OD021527, and DOE contract DE-AC02-06CH11357. We thank staff at Advanced Photon
Source beamline 24-ID-E for assistance in data collection and Y. V. Jiang for statistical
consultation and edits to the manuscript.

Author contributions J.S. conceptualized the project, expressed and purified proteins,
performed crystallization, carried out protein pull-down experiments and the pseudovirus entry
assay, and reviewed the manuscript. G.Y. performed crystallization, determined and refined the
structure, analysed the structure, performed the SPR experiment, and reviewed the manuscript.
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